Horseradish contains many bioactive compounds with antioxidant activity. The current study aimed to evaluate the effect of various wall materials and their ratios on the physical properties and bioactive-compound retention and stability in microencapsulated horseradish leaf and root juices. Horseradish juice was microencapsulated using maltodextrin, maltodextrin/gum Arabic, soy protein isolate, and starch with three different core-to-wall ratios. The total phenolic, total flavonoid, total flavan-3-ol, and total phenolic-acid contents, as well as antioxidant activity, were determined using spectrophotometric methods, whereas individual phenol profiles were determined by high-performance liquid chromatography (HPLC). Multivariate analysis of variance showed that plant material, wall material, and core-to-wall ratio had a significant effect on the bioactive-compound retention and antioxidant-activity preservation. Microcapsules produced from horseradish leaf juice had a significantly higher content of phenolic compounds and antioxidant activity compared to root-juice microcapsules. However, better retention was observed for microencapsulated horseradish root juice. Maltodextrin and maltodextrin/gum Arabic were the most effective wall materials for the retention of bioactive compounds, while they also had a smaller particle size and better solubility. The horseradish-juice microcapsules possess a high content of rutin. The highest stability of bioactive compounds after storage was found at a core-to-wall ratio of 20:80. Armoracia rusticana L.) is a perennial herb of the Brassicaceae family, widely used in culinary and folk medicine. A recent study by Dekic et al. [Horseradish , indicating the importance of the wall-to-core ratio. However, efficiency is highly dependent on the food matrix, the type of phenolic compound encapsulated, and the coating composition [A large variety of materials can be used for the encapsulation purposes . Polysacposition .The size and distribution of particles obtained in the spray-drying process vary in a wide range ,13. TheyThe current study aimed to evaluate effect of various wall materials and their ratios on the physical properties and bioactive-compound retention and stability in microencapsulated horseradish leaf and root juice. The total phenolic compounds, total phenolic acids, total flavonoids, and total flavan-3-ols, as well as antioxidant activity, were studied.Armoracia rusticana L.), which was used for juice production, was collected in Latvia ; the leaves were collected in July 2018, while the roots were collected in November 2018. Plant material was frozen and stored at −18 °C until further use. Horseradish root and leaf juices were obtained by grinding the frozen samples before extraction in a basket press. The obtained juice was freeze-dried to 93% dry matter and used in further studies.Fresh horseradish  with a dextrose-equivalent value (DE) of 12, gum Arabic (G), and soluble starch (S), were purchased from Sigma-Aldrich . Soy protein isolate (SPI), 90% pure, was purchased from Lustrel Laboratoires SAS .•) were purchased from Sigma-Aldrich . All other chemicals used in the research were obtained from Acros Organics .All chemicals were of analytical or high-performance liquid chromatography (HPLC) grade. Gallic acid, Folin–Ciocalteu phenol reagent, and 2,2-diphenyl-1-picrylhydrazyl (DPPHw/w) total solids.Different core-to-wall ratios of 20:80, 50:50, and 80:20 with various wall materials were prepared for the study . The infThe wall material and freeze-dried juice were dissolved in deionized water.Maltodextrin and maltodextrin/gum Arabic with juice were mixed at room temperature for 30 min under constant mechanical stirring (1000 rpm). Soy protein isolate with juice was stirred for 30 min at 70 ± 2 °C at 5000 rpm and further held in the same conditions during spray-drying.Starch was dissolved in deionized water at room temperature for 30 min under mechanical stirring at 1000 rpm; then, after emulsion, it was heated at 90 ± 2 °C for 30 min under mechanical stirring (1000 rpm), in order to allow maximum starch solubilization. Then, the starch solution was left overnight at room temperature. The next day, the starch solution with juice was mechanically stirred (1000 rpm) at room temperature for 30 min.The total amount of prepared solution for each sample was 500 g; each experiment was performed in triplicate.Freshly prepared infeed solutions were spray-dried in a Mini Spray Dryer B-290  under the following stable process conditions: inlet air temperature, 120 ± 4 °C; outlet air temperature, 80 ± 4 °C; drying airflow rate, 470 L/h; liquid feed flow rate, 0.33 L/h; aspiration, 100%. During the spray-drying process, solutions were mixed with a magnetic stirrer (magnet size 4.0 × 0.5 cm) at 500 rpm at room temperature (20 ± 1 °C), except for the solutions with SPI. All infeed solutions with SPI during the spray-drying process were mixed with a magnetic stirrer (magnet size 4.0 × 0.5 cm) at 500 rpm at 70 ± 1 °C. Microcapsules were collected in the cyclone collector, then sealed in hermetic packaging, and stored for further analysis in the dark at room temperature.Spray-dried juices without added materials were used as the control (blank).The microcapsule size in dry experimental powder was determined on the basis of the scattering pattern of a transverse laser light using Scirocco 2000 equipment . The mean particle diameters were determined ranging from 0.2 to 2000 μm. The used refractive index was 1.52, the air pressure of dispersion was 4 bars, and the degree of vibration was 70%. The volume-based particle diameter was calculated as the mean of three measurements per sample.For hygroscopicity measurements, 1 g of powder was placed in a container at 25 °C with a saturated NaCl solution, obtaining 75.29% relative air humidity ,15. SampSolubility was determined according to the method proposed by Cano-Chauca et al. , with soThe RE of each biologically active compound was calculated using Equation (1).Microencapsulated horseradish-juice powders were packaged in hermetically sealed plastic containers and stored at room temperature (22 ± 1 °C) in the dark. The stability of biologically active compounds after four months of storage was calculated using Equation (2).For all samples, the moisture content was determined in triplicate according to the standard ISO 6496:1999, and all results were expressed on the basis of dry weight (DW).For the extraction procedure, 0.1 g of powder was dissolved in 100 mL of distilled water with the assistance of a magnetic stirrer (magnet size 4.0 × 0.5 cm) at 700 rpm for 20 min at room temperature (20 ± 1 °C). Spectrophotometric analysis was completed using a JENWAY 6300 .Total phenolic content (TPC) was determined with the Folin–Ciocalteu spectrophotometric assay  in mg ofTotal phenolic-acid content (TPAC) was determined using a spectrophotometric method , expressTotal flavonoid content (TFC) was determined according to a colorimetric method  with modTotal flavan-3-ol content (TF3C) content was determined using Zam et al.’s  method, •) radical-scavenging activities were determined according Yu et al.’s method [+•) radical cation assay, described by Re et al. [The 2,2-diphenyl-1-picrylhydrazyl  in an ultrasonic bath YJ5120-1  at 35 kHz for 10 min at 20 ± 1 °C. The extracts were then centrifuged in a CM-6MT centrifuge  at 3500 rpm for 5 min. The supernatants were combined in a 25 mL graduated flask and filled to the mark with solvent. Separation and quantification were carried out using a Prominence high-performance liquid chromatography system LC-20AD  with a YMC C18 analytical column and a SPD M20A photodiode array detector according to the procedure described by Priecina et al. [The weight of the microencapsulated samples for extraction was selected to obtain equal dry-matter mass; 0.25 ± 0.01 g of sample was extracted with 25 mL of 1 N HCl/EtOH/Hp ≤ 0.05. Multivariate analysis of variance (MANOVA) was used to evaluate the influence of factors such as plant part, wall material, and core-to-wall ratio on the dependent variables . Estimated marginal means for each factor were presented. A linear correlation analysis was carried out using SPSS version 17 .The results are presented as the mean value ± standard deviation. Analysis of variance (ANOVA) and Tukey’s multiple-comparison test were carried out to determine differences in parameters among the samples. Differences were considered as significant at The particle size of the microcapsules have a significant effect on sensory properties  and physical properties . The particle size of the obtained microcapsules ranged from 3.64 µm (R50MG50) to 18.40 µm (R20S80) . This isThe size of the microcapsules was significantly affected by the core material; the horseradish leaf microcapsules were on average 12% larger compared to the root microcapsules. The wall material significantly affected the particle size, whereby samples obtained with maltodextrin (MD) and maltodextrin/gum Arabic (MG) had smaller particles compared to starch (S) and soy protein isolate (SPI) microcapsules.p < 0.05) influenced the particle size of the horseradish-juice microcapsules. Rodsamran et al. [The core-to-wall ratio significantly  to 20.89 g/100 g DW (R80M20) . There wp < 0.05) effect on the hygroscopicity of horseradish-juice microcapsules. According to Ferrari et al. [The wall material had a significant  effect on the hygroscopicity of horseradish-juice microcapsules. The results of the study showed a tendency of the hygroscopicity of microencapsulated horseradish juice increasing as the proportion of wall material decreased. De Souza and colleagues made the same observations [r = −0.822; for root juice, r = −0.606).The concentration of the wall material or the wall thickness also affects the hygroscopicity of the microcapsules . The corrvations . In our p < 0.05) affected by both the wall material and the core-to-wall ratio (p < 0.05) lower solubility was observed for microcapsules with starch. This was consistent with the findings for particle size, where microcapsules with larger particle sizes (such as those using starch (S) and soy protein isolate (SPI)) had lower solubility.Solubility is one of the most important functional properties of edible powders. It affects the behavior of the powder when it is reconstituted in water. The solubility of horseradish-juice microcapsules was significantly  pulp and residue with GA and MD (DE 9–12) was 99.0%–99.1%, as explained by the properties of wall material used and by the particle size obtained. Specifically, a smaller particle size leads to a larger available surface area for the binding of water molecules, i.e., hydration [r = −0.874) and horseradish roots (r = −0.921). A moderate correlation was also established between solubility and hygroscopicity .Very good water solubility of microcapsules was also observed by Rezende et al. , where tydration . CorrelaAlthough there were scientists  who founIn addition, microcapsules with starch and SPI tended to increase the solubility as the proportion of core material increased. In contrast, microcapsules with MD and MG showed the opposite tendency, whereby the solubility decreased with an increasing proportion of core material. This can be explained by the fact that starch has low solubility and high viscosity, whereas MD has high solubility and low viscosity. In this study, the lowest solubility was seen in horseradish-juice microcapsules with SPI, which is similar to the findings of Molina Ortiz et al. . However•-scavenging activity was confirmed for the horseradish-leaf-juice powder samples encapsulated using the SPI, MG or S as the wall material, with a core-to-wall ratio of 80:20. The highest ABTS•+ activity was observed for samples L80P20 and L80S20. Horseradish-root-juice sample R80MG20 had the best reducing power among the studied microcapsule samples.The content of phenolic compounds and the antioxidant activity (AOA) of the horseradish-root-juice and horseradish-leaf-juice microcapsules obtained by spray-drying are presented in Similar studies on plant-extract encapsulation proved that the AOA in microencapsulated garlic extract was between 1.15 ± 0.01 and 1.44 ± 0.02 mmol TE/100 g DW , the AOAp < 0.05) on the TPC, TPAC, TFC, TF3C, and AOA on the basis of the three tested methods, namely, DPPH•, ABTS+•, and RP.Multivariate analysis of variance showed that plant material (roots and leaves), wall material , and core-to-wall ratio had a significant effect (p < 0.05) higher content of phenolic compounds and AOA compared to the root-juice microcapsules  due to the included proportion of wall material, which did not contain phenolics, except for the sample with soy protein isolate.Nephelium lappaceum L.) peel extract. According to Pang et al. [Orthosiphon stamineus extract was effective, ranging from 66.91% (eupatorin) to 82.08% (rosmarinic acid); however, during microencapsulation by spray-drying, phenolic compounds were lost due to high temperature and the action of oxygen, resulting in their destruction and polymerization [For the evaluation of the microencapsulation process, the retention of compounds in the final product is a very important factor. In our study, the average retention coefficients were higher for root samples , except g et al. , the retrization . DespiteIn general, better retention was observed with microencapsulation of horseradish root juice . This coThe retention of rutin was highest in the root juice, especially when using maltodextrin in combination with gum Arabic as the juice wall material b. The op•- and ABTS+•-scavenging activity. MD is the most popular and widely used natural material for microencapsulation. It is a polysaccharide, making it very convenient for use in the microencapsulation process by spray-drying due to its good water solubility. According to Kuck and Noreña [Murraya koenigii L. leaf extract confirmed MD as the least effective wall material for phenolic-compound retention compared to xanthan and acacia gums [The type of wall material significantly affected the RE . Specifid Noreña , polysaccia gums . In the The application of maltodextrin combined with gum Arabic resulted in better retention of TPC, TPAC, and TFC, and better RP. These compounds were retained more efficiently because the use of a higher-molecular-weight coating material increased the rate of dry-film formation on the surface of the microcapsule . In a miMoringa oleifera leaf extract in terms of TPC, its efficiency was found to range from 52% to 75% [Soy protein isolate did not demonstrate the best retention for any of the tested parameters, but it proved to be more effective for preserving TF3C and TFC. SPI has several drawbacks with regard to its application for spray-drying due to its low water solubility and high losses in the drying process. When analyzing the efficacy of SPI for the encapsulation of % to 75% .Hibiscus sabdariffa L.Soluble starch did not achieve the best retention for any of the tested parameters, but higher results for the retention of TPC and TFC were observed. Similar to SPI, soluble starch has several drawbacks for use in the spray-drying process, due to its difficulty in forming a homogeneous suspension compared to the other wall materials analyzed. In the study of Idham et al. , solubleEuterpe edulis M.) juice and maltodextrin microcapsules (1229.4 mg GAE/100 g) [The combination of MD and GA as a coating material yielded horseradish-root-juice microcapsules with the highest RP and the highest (+)-catechin content, as well as horseradish-leaf-juice microcapsules with the highest TPAC and higher ABTS-cation-scavenging activity . FurtherE/100 g) , garlic E/100 g) , and redE/100 g) .for horseradish leaf juice, MG > M > S > SP;for horseradish root juice, M > MG > SP > S.For encapsulation, the wall materials could be ranked in order of their effectiveness in retaining bioactive compounds (starting with the most efficient):p < 0.05) , even lower stability than the blank sample was observed. Overall, a core-to-wall ratio of 80:20 was not suitable for the encapsulation of horseradish products. Osamede, Airouyuwa, and Kaewmanee [The effect of core-to-wall ratio between plant extract and wall materials in the encapsulation process is a very important factor, significantly affecting the stability of microcapsules during their storage ( < 0.05) . A loweraewmanee  tested tThe highest stability of TPC, TFC, and TF3C was achieved in the samples encapsulated using MG, reaching an estimated marginal mean for stability of up to 95% for TPC after four months of storage . In contThe degradation of some biologically active compounds during storage can be attributed to the presence of oxygen in the powders and a higher water activity, leading to a higher rate of decomposition .The plant part, wall material, and core-to-wall ratio significantly affected the physical parameters of the horseradish-juice microcapsules. Maltodextrin (MD) and maltodextrin/gum Arabic (MG) microcapsules had smaller particles and better solubility but displayed higher hygroscopicity. On the other hand, microcapsules with starch had lower hygroscopicity and solubility but a larger particle size.The results of this study confirmed the efficiency of microencapsulation for preserving bioactive compounds. However, the plant material, wall material, and core-to-wall ratio significantly affected the retention and stability of the bioactive compounds and the antioxidant activities of microcapsules during their storage. Despite the horseradish-leaf-juice microcapsules containing more bioactive compounds, better retention was seen in the horseradish-root-juice microcapsules. Among the wall materials studied, maltodextrin and maltodextrin/gum Arabic were more effective for the retention of bioactive compounds. In addition, the core-to-wall ratio of 20:80 was more stable during storage. However, more research is required to evaluate the potential application of these microcapsules in food.


Whether the subtype of atrial fibrillation affects outcomes after transcatheter aortic valve replacement for aortic stenosis is unclear. The nationwide FinnValve registry included 2130 patients who underwent primary after transcatheter aortic valve replacement for aortic stenosis during 2008–2017. Altogether, 281 (13.2%) patients had pre-existing paroxysmal atrial fibrillation, 651 (30.6%) had pre-existing non-paroxysmal atrial fibrillation and 160 (7.5%) were diagnosed with new-onset atrial fibrillation during the index hospitalization. The median follow-up was 2.4 (interquartile range: 1.6–3.8) years. Paroxysmal atrial fibrillation did not affect 30-day or overall mortality . Non-paroxysmal atrial fibrillation demonstrated an increased risk of overall mortality , but not 30-day mortality (p = 0.084). New-onset atrial fibrillation demonstrated significantly increased 30-day mortality  and overall mortality . The incidence of early or late stroke did not differ between atrial fibrillation subtypes . In conclusion, non-paroxysmal atrial fibrillation and new-onset atrial fibrillation are associated with increased mortality after transcatheter aortic valve replacement for aortic stenosis, whereas paroxysmal atrial fibrillation has no effect on mortality. These findings suggest that non-paroxysmal atrial fibrillation rather than paroxysmal atrial fibrillation may be associated with structural cardiac damage which is of prognostic significance in patients with aortic stenosis undergoing transcatheter aortic valve replacement. Transcatheter aortic valve replacement (TAVR) has demonstrated its efficacy in the treatment of severe symptomatic aortic stenosis (AS) among patients who are inoperable or at a high or moderate surgical risk , 2. AtriThe FinnValve registry is a Finnish multicenter nationwide registry including retrospectively collected data from consecutive patients undergoing TAVR or surgical aortic valve replacement with a bioprosthesis for severe AS at all five Finnish university hospitals  between January 2008 and November 2017  . The stuAll patients over the age of 18 years with AS undergoing TAVR or surgical aortic valve replacement with a bioprosthesis during the study period meeting the inclusion criteria were included in the registry . The excThe diagnosis and subtype of atrial fibrillation was determined from the clinical records of the study patients undergoing TAVR. Patients were classified in to four categories according to presence and subtype of AF: pre-existing paroxysmal AF, pre-existing non-paroxysmal AF, new-onset AF and no AF. Non-paroxysmal AF included chronic and persistent AF. New-onset AF was defined as AF diagnosed during the index hospitalization at the operative center. Among the patients undergoing TAVR, 281 (13.2%) patients had pre-existing paroxysmal AF, 651 (30.6%) patients had pre-existing non-paroxysmal AF and 160 (7.2%) patients were diagnosed with new-onset AF, while 1038 (48.7%) patients had no history of AF at the time of hospital discharge.th, 2019. Follow-up was considered complete for all patients except for those not residing in Finland, and their follow-up was truncated at hospital discharge.Data on baseline and operative characteristics and postoperative outcomes were retrospectively collected in a standardized online electronic data collection form. Data on the date of death were obtained from the Population Register Center, a governmental agency, which reviews all death certificates issued in Finland. Data on cardiovascular interventions were obtained from the Finnish National Institute for Health and Welfare registry, which collects information on the diagnosis and treatment of patients admitted to any local, regional or tertiary hospital. The last day of follow-up was January 26Baseline variables were defined according to the EuroSCORE II criteria . CoronarThe primary outcome measure of the current study was all-cause overall mortality. The secondary outcome measures were all-cause 30-day mortality and 5-year mortality, early stroke occurring during the index hospitalization, late stroke occurring after discharge as well as combined early or late stroke. Outcome analyses were not landmarked.https://www.R-project.org). The normality assumption of continuous variables was assessed visually and with the Kolmogorov-Smirnov test of normality. Continuous data are presented as median (interquartile range) and categorical variables as absolute number and percentage. The Fisher’s exact test and the chi-square test were used to compare differences between proportions, and the Kruskal-Wallis test and the Mann-Whitney U-test to analyze continuous variables. The Kaplan-Meier method was used to assess survival. Differences in survival between patients with paroxysmal, non-paroxysmal or new-onset AF and those without AF were evaluated with the log-rank test. A stepwise Cox proportional-hazards model with the Wald backward method was used to determine the adjusted hazard of overall, 30-day and 5-year mortality mortality associated with AF. All variables with p<0.1 in univariate Cox regression analyses were included in to the multiple Cox model. The proportional hazards assumption was ensured by assessing the test based on Schoenfeld residuals, as well as by graphical assessment of the ln-ln plot of the survival curves. Binary logistic regression models with the Wald backward method were used to define the adjusted odds ratio of early, late or any stroke during the follow-up. A binary logistic regression analysis with the Wald backward method was also performed to assess the independent predictors of new-onset AF. The goodness of fit of the binary logistic regression models were estimated by the Hosmer-Lemeshow's test. All variables with p<0.1 in univariate analyses were included in to the logistic regression models. Two-sided differences were considered significant if the null hypothesis could be rejected at the 0.05 probability level.Statistical analyses were performed with the IBM SPSS Statistics software  and R software  years and 958 (45.0%) were men. The baseline and operative characteristics of the patients are presented in During the follow-up (median 2.4 (1.6–3.89 years), the primary outcome measure of all-cause mortality occurred in 85 (30.2%) patients with paroxysmal AF, 265 (40.7%) patients with non-paroxysmal AF and 71 (44.4%) patients with new-onset AF. The median survival time was 4.6 (2.2–7.0) years in patients with non-paroxysmal AF, 5.2 (2.7–9.0) years in patients with paroxysmal AF, 4.5 (3.2–5.1) years in patients with new-onset AF and 6.6 (3.6–8.6) years in those with no AF (log-rank <0.001). The hazard ratio (HR) of all-cause mortality was 1.36 : 1.06–1.74; p = 0.014) for paroxysmal AF, 1.81  for non-paroxysmal AF and 1.84  for new-onset AF as compared with the 259 (25.0%) patients without AF. Survival in patients with paroxysmal, non-paroxysmal, new-onset and no AF before TAVR are presented in The adjusted risk estimates of study outcomes are shown in Among the patients with no previous history of AF before the TAVR procedure, the independent predictors of new-onset AF were critical preoperative state , transapical access  and older than third generation prosthesis . Compared to patients who received a third generation TAVR prosthesis, moderate to severe paravalvular regurgitation was more common  and operative risk higher as assessed by the STS score (3.23 (2.35–4.57) vs. 4.20 (2.99–6.20), p<0.001) or EuroSCORE II (3.84 (2.43–6.39) vs. 6.20 (3.58–11.10), p<0.001) in patients who received an older generation prosthesis. However, older than third generation prosthesis remained a predictor of new-onset AF even after forcing moderate to severe paravalvular regurgitation (p = 0.039), STS score (p = 0.034) or EuroSCORE II (p = 0.027) into the multiple logistic regression model. Hospital stay was longer in patients with new-onset AF (7 (4–12) days) compared to those with no AF (4 (2–6) days), paroxysmal AF (5 (3–7) days) or non-paroxysmal AF (5 (3–7) days) (p<0.001).The antithrombotic therapies of the patients with AF at discharge are described in The current study documented that AF is a common condition among patients undergoing TAVR for severe AS with a prevalence of approximately 50%. A novel and relevant finding was that among patients with pre-existing AF undergoing TAVR, long-term mortality was significantly increased in those with a non-paroxysmal form of the arrhythmia, whereas paroxysmal AF had no effect on mortality. Secondly, new-onset AF was predicted by critical preoperative state, transapical access technique and use of older generation TAVR prostheses and was associated with both increased short and long-term mortality after TAVR. Incidence of stroke during short or long-term follow-up after TAVR was unaffected by the subtype of AF.AF is a well-known predictor of mortality in the general population and in those with AS –18. ReseThe rate of new-onset AF in the current study was 7.2%, a finding in agreement with earlier studies –6. SomewTransapical access was found to be an important predictor of new-onset AF as shown also by a previous pooled analysis . The cauA novel and interesting finding of our study was that older generation TAVR prostheses were an independent predictor of new-onset AF. Older generations of TAVR prostheses are associated with a distinctly higher rate of paravalvular regurgitation , 25. SigAF is a major risk factor of thromboembolism and stroke, and stroke is in turn a common severe adverse event after TAVR , 27. HowThe optimal antithrombotic management after TAVR in patients with AF is unclear. Current guidelines recommend the use of a vitamin K antagonist either alone or in combination with an antiplatelet agent for 3 to 6 months , 2. HoweThe present findings are of clinical importance because they suggest that non-paroxysmal AF, but not paroxysmal AF, is associated with structural changes of the atrial wall which have prognostic importance. Fibrosis of the atrial wall is an established trigger for AF and a recent study showed that focal left atrial fibrosis localized to the left inferior pulmonary vein antrum, the extent of left atrial fibrosis, and enlarged left atrial volume were independently associated with persistent atrial fibrillation rather than with paroxysmal atrial fibrillation in cardiac MRI , 32. TheThere are some limitations to the current study that must be acknowledged. The main limitation is the retrospective design of the study with all its inherent challenges. New-onset AF was defined as being diagnosed during the index hospitalization for the TAVR procedure, the duration of which was variable, which may be seen as a limitation. By extending the period of post-operative detection, it is conceivable that the number of procedure-related new-onset AF cases could have been increased. Additionally, no routine electrocardiographic monitoring was performed prior to the TAVR procedure and it is therefore possible that some patients who were classified as having new-onset AF actually had undetected paroxysmal AF. Data on whether patients with new-onset AF had a paroxysmal or a non-paroxysmal form of the arrhythmia was not available, which is another limitation. A notable limitation is that the antithrombotic medication regimes between patients were not randomized and were rather based on individual risk assessment. Moreover, antithrombotic medications were recorded at admittance and discharge only, and possible regimen changes afterwards could not be accounted for. Lack of bleeding data is another limitation. On the other hand, these data represent a large unselected nationwide cohort of patients undergoing TAVR with a relatively lengthy follow-up strengthening the validity of the results. A particular strength of the study was the accuracy and completeness of the survival data.In conclusion, among patients undergoing TAVR, pre-existing non-paroxysmal AF and new-onset AF are associated with increased mortality, whereas pre-existing paroxysmal AF is not. These findings suggest that non-paroxysmal AF rather than paroxysmal AF may be associated with structural cardiac damage which is of prognostic significance. Further large-scale studies are needed to confirm these observations.


Escherichia coli and Bacillus subtilis; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in B. subtilis like it is in E. coli. In B. subtilis, RecA is essential to the process of genetic transformation in the subpopulation of cells that become naturally competent in conditions of stress. Interestingly, the transcriptional regulator ComK, which controls the development of competence, does influence the accumulation of mutations in stationary phase in B. subtilis. Since recombination is not involved in this process even though ComK is, we investigated if the development of a subpopulation (K-cells) could be involved in stationary-phase mutagenesis. Using genetic knockout strains and a point-mutation reversion system, we investigated the effects of ComK, ComEA (a protein involved in DNA transport during transformation), and oxidative damage on stationary-phase mutagenesis. We found that stationary-phase revertants were more likely to have undergone the development of competence than the background of non-revertant cells, mutations accumulated independently of DNA uptake, and the presence of exogenous oxidants potentiated mutagenesis in K-cells. Therefore, the development of the K-state creates conditions favorable to an increase in the genetic diversity of the population not only through exogenous DNA uptake but also through stationary-phase mutagenesis.Bacterial cells develop mutations in the absence of cellular division through a process known as stationary-phase or stress-induced mutagenesis. This phenomenon has been studied in a few bacterial models, including  Escherichia coli. One interesting question in the stationary-phase mutagenesis field is how a bacterial population manages increases in genetic diversity without increasing the chance for genetic load. In this regard, research has examined two aspects: (1) whether all the cells in a population are subject to mutagenesis, and (2) whether all the genome is subject to mutagenesis. In E. coli, stressed cells have evolved mechanisms to license the increase of error-prone repair at genetic regions surrounding DNA double-stranded breaks [−3 frequency spontaneously [E. coli manage increases in genetic diversity by activating mechanisms that operate on a subpopulation of cells and transcribed DNA undergoing repair of double-stranded breaks [For over 60 years, genetic experiments have shown that bacterial cells can develop mutations in the absence of growth; this is known as stationary-phase mutagenesis or stress-induced mutagenesis ,2,3. Thid breaks , which oaneously . Repair aneously ,8. Interaneously . Also, faneously . Therefoaspects:  whether B. subtilis halts replication and differentiates subpopulations [Bacillus subtilis also expresses stationary-phase mutagenesis. In these processes, the transcription repair coupling factor Mfd plays a central role, interacting with components of the nucleotide excision repair (NER) or base excision repair (BER) pathways and error-prone polymerases to produce mutations in stationary-phase cells [In the context of increasing genetic diversity in times of nutritional stress, ulations ,12. One ulations . Interesse cells . This ty+ reversions [+ reversions in stationary-phase B. subtilis are promoted by the late steps that differentiate cells into competence in the absence of genetic recombination.Sung and Yasbin contributed two interesting observations to our understanding of stationary-phase mutagenesis . RecA, aversions . These oCharacterization of the formation of the competent subpopulation has redefined this cell differentiation pathway as a mechanism to cope with stress that goes beyond promoting genetic recombination . Transcr+ mutagenesis occurs in K-cells that are deficient in the uptake of DNA, but at slightly lower levels than in cells containing a functional uptake apparatus (ComEA+ cells). To better study this phenomenon, we used a ComK-inducible system to turn all the cells in the culture into the K-state and measure mutagenesis. We found that ComEA− cells produced fewer stationary-phase mutants than their ComEA+ counterparts but tolerated oxidant exposure better than ComEA+ cells. Furthermore, K-cells showed better survival to oxidative stress than non-K-cells, which indicated that this differentiation state activates mechanisms that repair oxidative damage. We followed these observations with microscopy assays of K-cells differing in ComEA and measured fluorescence of an indicator dye as a proxy for oxidative damage. These assays showed that, under oxidative stress, ComEA− cells were less damaged than ComEA+ cells. These results support the concept that developing into the K-state, which includes installing a functional DNA uptake system in the cell surface, predisposes B. subtilis to oxidative damage. K-cells activate mechanisms to survive oxidative damage and increase mutagenesis in cells under non-lethal selection pressure. Therefore, B. subtilis K-cells increase genetic diversity via recombination-dependent and recombination-independent pathways. Our experiments showed that cells that undergo genetic transformation are more likely to become stationary-phase mutants and that oxidative damage to DNA is a precursor to the formation of mutations. Furthermore, stationary-phase MetB. subtilis strains used in this study are all derivatives of the strain YB955 and are described in hisC952, metB5, and leuC427 [B. subtilis strains were all maintained on tryptose blood agar base medium  or grown in liquid cultures in Penassay broth (PAB)  with antibiotics, such as 5 μg/mL of neomycin (Nm), 100 μg/mL of spectinomycin (Sp), 5 μg/mL chloramphenicol (Cm), or 10 μg/mL of tetracycline (Tc), as needed. Escherichia coli strains were maintained and grown on Luria–Bertani (LB) with antibiotics as needed.comEA gene (JC101) was constructed by cloning a neomycin cassette within the comEA gene. Two sets of primers . Transformants were selected on ampicillin and screened by restriction enzyme analysis. This plasmid was transformed into YB955, as previously described [B. subtilis YB955 was grown to T90, ninety minutes after the cessation of growth (stationary phase), in GM1 broth . After one hour of incubation at 37 °C with aeration, plasmid DNA (100 ng) was added. The culture was incubated for another hour, followed by the addition of 100 μL of 10% yeast extract. After an additional hour of incubation, the cells were plated on TBAB containing 5 μg/mL neomycin to select for the RcomEA::neo allele. Then -comEA transformants were screened by PCR.The strain containing a defective mers see  were despBest502 . The fraescribed . Brieflysolution  and thencomK-gfp IPTG-inducible strain, genomic DNA was isolated from B. subtilis BD401021 using the Wizard® Genomic DNA Purification Kit . Isolated genomic DNA was then transformed into YB955 using the competence procedures for B. subtilis described above [amyE::pHS-comK allele and 5 μg/mL chloramphenicol to select for the comK-gfp allele. Transformants were verified by flow cytometry using IPTG and comparing the fluorescence intensity of induced versus uninduced cells in relation to parental cells (YB955).To construct the ed above . Cells wgfp-comK IPTG-inducible comEA knockout strain, genomic DNA from JC101 was extracted using Wizard® Genomic DNA Purification Kit . The genomic DNA was then transformed into HAM501 using the competence protocol described above. Cells were plated on TBAB containing 100 μg/mL spectinomycin, 5 μg/mL chloramphenicol, and 5 μg/mL neomycin. Transformants were verified using the transformation assay described above and transformed using the plasmid pDG1664 [To construct the  pDG1664  containi2, one colony of each strain was used to inoculate two mL of PAB. This was grown overnight in a shaking incubator at 37 °C with aeration (250 rpm). The next day, 1 mL of the overnight culture was transferred into 20 mL of PAB with 20 µL of 1000× Ho-Le trace elements into a 250-mL Erlenmeyer flask. Growth was monitored with a Genesys 10S UV–vis  until T90. Cells were then harvested by centrifugation at room temperature and resuspended in 1× Spizizen minimal salts. Cells were then plated in quintuplet (100 µL per plate) on 1× Spizizen minimal medium , they were treated with 0 or 1.5 mM tert-butyl hydroperoxide  Sigma-Aldrich, St. Louis, MO, USA) for two hours, washed with 1X SMS twice, then plated, and assayed for stationary-phase mutagenesis.To determine the effects of oxidative stress on 90. The culture was then diluted 10-fold into GM2 and incubated for one hour as previously described. Finally, the culture was split into two different batches. Then, 150 ng of the transforming DNA  was added to one subset of the culture, while nothing was added to the other subset of the culture, which served as the no-DNA control. In addition, a broth-only control was used.Strains were grown overnight in GM1 with Ho-Le trace elements in a 250 mL Erlenmeyer flask on a bench top at room temperature for approximately 16 hours. The next day, the culture was incubated in a shaker at 37 °C with aeration (250 rpm). Growth was monitored with a Genesys 10S UV–vis  until T10 units and plated onto medium containing all the required amino acids. The experiments were repeated at least three times. The viability of the cells of the non-revertant background cells was assessed as described above.Cells were incubated for an additional hour, followed by the addition of 10% yeast extract to all three subsets. Cells were incubated one final hour and subsequently harvested. Cells were harvested by centrifugation at room temperature and resuspended in 1× SMS. Cells from the DNA treatment and no DNA cultures were plated in quintuplet (100 μL per plate) on both SMM and TBAB with 100 ng/mL of spectinomycin, which assessed transformation efficiency and spontaneous spectinomycin reversion. The SMM plates were incubated for 9 days with revertants scored every day. To determine the initial number of cells plated, the beginning cell suspension was serially diluted by 6 logTo determine if prototrophic revertants were also transformants, colonies were screened for growth on TBAB with 100 ng/mL of spectinomycin. As a control, non-revertant colonies  were subject to the same screening.B. subtilis strains, HAM501 and AAK502, were grown to mid-exponential phase and T90. A batch of cells were divided into two sets at a late exponential phase (0.7–0.8 OD600) and IPTG was added to one set. Then, both sets (with and without IPTG) were grown until T90 (stationary phase). A 1-mL aliquot from each condition was removed at a late exponential phase, as well as the stationary phase, and combined with 50 μL of 37% formaldehyde. This mixture was centrifuged for 10 min at 300× g (2100 rpm) in a microcentrifuge tube. The supernatant was decanted, and the pellet was resuspended in 1 mL of phosphate-buffered saline (PBS). Samples were covered in foil until they were run through the BD FACSCalibur flow cytometer.600 of approximately 0.75. Then cultures were divided into halves, and one half was induced with 1 mM IPTG. We monitored the growth of the cultures until the transition from exponential to stationary phase was identified . At T50 (50 minutes after the cessation of growth), each condition was pre-labeled by adding 5 μM peroxy-orange 1 . PO1 measures the concentration of H2O2 since PO1 only becomes fluorescent after oxidation by H2O2 which removes a protective boronate side group [t-BHP. This technique has been used to measure ROS in different cell types [90. At this point, each condition was divided and exposed to either 0 or 5 mM of t-BHP for 2 hours. Cells were then harvested by centrifugation, washed twice with 1X SMS, and serially diluted. One colony of each strain was used to inoculate 2 mL of PAB. This culture was grown overnight in a shaking incubator at 37 °C with aeration (250 rpm). One milliliter of the overnight culture was transferred into 20 mL of PAB with 20 µL of 1000× Ho-Le trace elements into 250 mL Erlenmeyer flask. Growth was monitored with a Genesys 10S UV–vis  until ODde group . Therefoll types ,23. CultTo determine percent survival, we plated 0.1 mL of serial 10-fold dilutions onto TBAB, incubated the plates for 24 hours at 37 °C, and scored for colonies to determine survival. Percent survival was determined by dividing the number of CFUs from the exposed conditions by the number of CFUs from the 0 mM condition. We repeated this procedure at least three times, with three replicates each time.−1 diluted culture was immobilized in a 1% agarose pad on a clean glass slide before imaging. Live cell imaging was conducted using transmitted light and fluorescence microscopy equipped with oil immersion (Plan-Apochromat 63×/1.40 Oil Ph3 M27). Fluorescence excitation was performed (X-cite 120LED) at a range of 550–605 nm (43 HE dsRed Zeiss Filter). Images were captured by an ORCA Flash 4.0 LT Monochromatic Digital CMOS camera. Image analysis was completed using MicrobeJ. Fluorescent foci were detected and counted using the maxima feature in MicrobeJ. Maxima are considered the regions of highest fluorescence within a cell. We used this measurement to quantify the amount of oxidized PO1 molecules within cells. Cell length was measured using the medial axis feature in MicrobeJ [To visualize the amount of ROS in the cell, 0.8 μL of a 10MicrobeJ . At leas2 test. For the chi-square analysis, we first standardized the number of SpcR colonies for each population to the non-revertant cells that were transformed with DNA (n = 1755). Then, we constructed a contingency table to test the independence between the two conditions of transformation (DNA added and No DNA) and the population type  and found that these variables were independent . Then, we tested the number of transformants in each population type for each condition of transformation separately. In the DNA added condition, we used the number of transformants in the non-revertant population as the expected value; the number of transformants in each of the revertant populations (early and late revertants) was considered the observed value. The chi-square value for the DNA added condition was 208 (d.f. 1), which indicated a significant difference between the non-revertant and revertant populations. The same analysis was repeated in the No DNA added condition and the chi-square value was 3.6 (d.f. 1), which indicated no significant differences among the three populations. The results are presented in percent in We used SPSS 24 software for data processing and analysis. To test whether the proportion of stationary-phase revertants that underwent transformation was different from the proportion of non-revertants that underwent transformation, we conducted a chip ≤ 0.05. Means were tested using the LSD test at p ≤ 0.05. The data from experiments in which cells were untreated or treated with t-BHP was LOG transformed to normalize distributions and analyzed by ANOVA. We used a letter system to denote significant differences between means. We assigned “a” to the means that were not significantly different from the mean with the highest value, “b” to means that were different from the “a” group, and so on.For the rest of the experiments, statistical significance was determined by performing ANOVA. Significance was tested at recA and the general stress sigma factor σB (σS in E. coli). These genes encode key factors (RecA) or activate genes important in stress-induced mutagenesis in E. coli [+ reversions, but the inactivation of ComA, which controls the early steps, did not [+ reversions in stationary-phase B. subtilis are promoted by the late steps that differentiate cells into competence in the absence of genetic recombination. Experimentally, we modified our stationary-phase mutagenesis assay to measure the contribution of the development of the K-state among revertants. Briefly, cells of the strain YB955, which is auxotrophic for methionine, were grown to T90  and prepped for transformation. Then, the plasmid, pDR111, which contains a selectable spectinomycin cassette, was supplied to half of the cells. The spectinomycin cassette is not linked to any of the methionine auxotrophy. The other half of the cells underwent the same preparation but were not exposed to the plasmid. Instead of selecting for the transformation of the plasmid, we plated the cell preparations to select for Met+ stationary-phase colonies. We tracked revertant colonies for nine days but considered only those colonies that arose on day 5 or later to be stationary-phase mutants  containing 100 μg/mL spectinomycin, which selects for transformants . When wemutation . To esticomEA gene, and measured stationary-phase Met+ mutagenesis. The ComEA protein is a component of the competence pore that binds and uptakes DNA during the transformation of B. subtilis [− strain is transformation-defective, as previously reported  cells were grown to T90, induced into the K-state by supplying IPTG, exposed to 0- or 1.5-mM t-BHP for two hours, washed twice, and plated on minimal medium lacking methionine. The results over the nine days are presented in t-BHP. However, in the absence of t-BHP treatment, cells lacking ComEA accumulated fewer mutants than ComEA+ cells. Strikingly, when K-cells were damaged with t-BHP, ComEA+ cells accumulated significantly more mutations than those that lack ComEA (−) to revert to the revertant levels of HAM501 (ComEA+) in conditions in which cells were not treated with t-BHP (Recent work has demonstrated the importance of reactive oxygen species (ROS) in the generation of stationary-phase mutations . Interesck ComEA A. The back ComEA , and thoth t-BHP A indicatt-BHP and in conditions in which cultures were non-induced or induced into the K-state (addition of IPTG). Survival assays indicated that tolerance to oxidative damage was increased in K-cells compared to non-K-cells  predisposes the cell to cytotoxic and genotoxic damage, which in turn activates a protective, error-prone DNA repair response that produces mutations. Interestingly, studies that examine protein networks and endogenous DNA damage revealed that increases in reactive oxygen species are associated with the presence of transmembrane transporters in E. coli [B. subtilis showed activation of detoxication mechanisms [Next, we tested the possibility that the uptake of exogenous DNA, another trademark characteristic of the K-state, influences stationary-phase mutagenesis. However, the DNA-uptake defective strain showed no significant differences in stationary-phase mutagenesis levels when compared to its wild-type counterpart. This result suggested that the process of DNA binding and uptake are dispensable to the formation of stationary-phase revertants . Interes E. coli . Furtherchanisms ,34.tert-butyl hydroperoxide. The results indicated the following. (1) In the absence of ComEA and t-BHP treatment, K-cells still produced stationary-phase mutants. (2) ComEA+ cells accumulated more mutants than cells lacking this protein. (3) When cells were exposed to an oxidant, the same response pattern was observed in stationary-phase mutagenesis  and pro[E. coli  and incr[E. coli .


The aim was to determine the final adult height (FAH) achieved by recombinant human growth hormone (rhGH) treatment, the factors affecting FAH and the success of attaining the genetic potential.Data of 133 patients treated with rhGH therapy were reviewed retrospectively. Patients were grouped according to diagnosis, either isolated GH deficiency (IGHD) or multiple pituitary hormone deficiency (MPHD), and by sex, and pubertal status at the beginning of treatment.The mean age of initiation of treatment was 12.3±2.18 years, and the mean duration of rhGH treatment was 3.65±1.5 years. The mean height standard deviation score (SDS) at diagnosis was -3.11±0.75 SD. All patients received a standardized GH dose of 0.033 mg/kg/day. Mean FAH-SDS was -1.8±0.77 and delta height-SDS (the change in height SDS between the beginning and end of treatment) was 1.28±0.94 SD. FAH-SDS was -1.79±0.86 SD in males; -1.82±0.64 in females (p=0.857); -1.94±0.71 at the beginning of treatment in pubertal patients and -1.68±0.81 in prepubertal patients (p=0.056); -1.84±0.89 in patients with IGHD and -0.47±0.2 in patients with MPHD (p˃0.05). In multiple regression analysis, First year delta height-SDS was the most predictive factor for both FAH-SDS and delta height-SDS.The majority of our patients achieved a final height compatible with their genetic potential as well as population standards when treated with rhGH even having started at a relatively late age. First year delta height-SDS was a predictive factor for FAH. Patients who the growth hormone (GH) treatment is started in the prepubertal period are known to achieve better final adult height (FAH) standard deviation score (SDS) and delta height SDS than those started in the pubertal period.The present study provides the data for height outcome of a large series of patients who reached the final height with GH therapy in a single center using a fixed dose GH therapy regimen. There was no difference between pubertal and prepubertal patients for FAH-SDS and delta height SDS with this fixed dose GH therapy. Commencement of GH therapy in the prepubertal period was not found to be associated with a better height outcome.The goal of growth hormone (GH) therapy is to increase the growth rate in the short term and to improve the final height which, in turn, affects the psychosocial status of the person in the long-term . GH alsoGH is administered subcutaneously with a dose of 0.025-0.1 mg/kg/day ,5 for siIn the present study, we recruited 133 patients with IGHD and MPHD who reached FAH from among the 557 patients who had received rhGH treatment in the Pediatric Endocrinology Department of Gazi Yaşargil Training and Research Hospital, a tertiary pediatric endocrine centre, between 2010 and 2018 . The stuth percentile for age and sex, a GH peak below 10 ng/mL on two GH provocation tests (clonidine and L-dopa), bone age retarded more than 2 SD in prepubertal patients and having open epiphysis in pubertal patients  was used for statistical analyses. Data were displayed as mean±SD or median [25-75 interquartile range (IQR)]. Kolmogorov-Smirnov and Shapiro-Wilk tests were used for normality distribution of the data. In order to compare the data, an independent sample t-test was used in the normally distributed groups, and non-parametric tests were used in the non-normally distributed groups. To evaluate the relationship between FAH-SDS and delta height SDS with PAH-SDS, MPH-SDS, first year delta height SDS, treatment duration, age and GHD diagnostic parameters  Pearson correlation analysis and multiple regression analysis was performed. A p value <0.05 was considered statistically significant.A total of 133 patients  were included in the study. At the beginning of the treatment, 63 (47.4%) of the patients were pubertal and 70 were prepubertal. In addition 123 (92.5%) had IGHD, and 10 (7.5%) had MPHD. Birth weight was lower than -2 SDS in 15 patients. Pretreatment mean height SDS was -3.11±0.75 and ranged from -6.1 to -1.71 SD. Height SDS was below -2 SD in all patients except for a patient with MPHD who had a height SDS of -1.71, who also suffered from recurrent hypoglycemia attributed to the GH deficiency which resolved after rhGH commencement. All patients with a height-adjusted weight below -2 SDS and/or BMI-SDS <-2 were also evaluated for protein-energy malnutrition. In our study, 85 of the patients were underweight according to height-adjusted weight SDS and 16 had BMI-SDS <-2. These patients were followed for at least a one year period with appropriate calorie intake. Patients who had low IGF SDS and low growth velocity in the first year of follow-up, despite appropriate calorie intake, were evaluated for GHD. Mean peak GH in the GH stimulation test was 5.32±2.41 ng/mL which ranged from 0.2 to 9.8 SD, mean IGF SDS was -1.2±1.04 and ranged from -3.4 to 2.1 SD. In total 80.4% of the patients were above the parent-specific lower limit for final height and 70% were within the normal range according to the population. Anthropometric measurements, laboratory values, and comparisons between the groups are shown in At the time of the treatment, age and bone age of pubertal patients were significantly higher than those of prepubertal patients (p<0.001) . There wGH peak, IGF1 SDS, IGFBP3 SDS, PAH-SDS were significantly lower in patients with MPHD compared to those with IGHD . There wIGF1 levels were below -2 SDS in 25 patients, initially. There was no statistically significant difference between the FAH-SDS of patients who had an IGF1 below or above -2 SDS. However, delta height SDS was higher in the group with an IGF1 below -2 SDS.FAH-SDS had a negative correlation with age and bone age at the beginning of treatment, and a positive correlation with height, weight, and first-year delta height SDS. Delta height SDS was negatively correlated with bone age, height, and weight, IGF SDS, IGFBP3 SDS, and positively correlated with first-year delta height SDS and treatment duration . In multIn the present study, the FAH achieved with GH therapy was evaluated and the factors affecting FAH in a group of patients who received 0.033 mg/kg/day GH treatment for IGHD and MPHD every day were investigated. Patients with MPHD had achieved a better final height (mean FAH-SDS was -0.47 SD) compared to cases with isolated IGHD (mean FAH-SDS was -1.84 SD).In studies conducted in our country, by Yordam et al  and KurnAlthough early diagnosis and treatment with GH can provide a final height consistent with MPH, patients’ final height usually remained below the average of the population ,17. SimiAbout half of our patients were at an age at which puberty would normally be expected to start when GH treatment was started. Although the chronological age and bone age were higher in the pubertal group and the duration of GH treatment was longer in the prepubertal group, there was no statistically significant difference between the two groups in terms of FAH-SDS and delta height SDS. Similar to our study, Kurnaz et al  did not In our study, PAH-SDS, weight SDS, and first year delta height SDS were predictive factors for both FAH-SDS and delta height SDS. Duration of treatment and bone age were the only predictive factors for FAH-SDS. Among these parameters, the most significant factor in predicting height gain at the end of the treatment was first-year delta height SDS. First year response of GH treatment, is one of the most important predictor of FAH ,26. DuriMPH-SDS has been reported as an important predictor of final height and patients with short parents have a poorer response to treatment . In our The present study has some limitations which may affect the FAH and delta FAH-SDS. About half of the 557 patients with GH deficiency and GH treatment who had been referred to our clinic from other centres but had become lost to regular follow-up. Therefore, the number of patients who reached FAH was low . MoreoveIn conclusion, the majority of our patients have achieved a final height compatible with their genetic potential and population standards even starting at a later age. First year delta height SDS was found to be the most predictive factor for FAH. Interestingly, the commencement of GH therapy in the prepubertal period was not found to be associated with a better height outcome. Recognition of these factors and individualization of the treatment accordingly will help to optimize the long-term response to GH treatment.